Immunoglobulins, or antibodies, are a group of molecules that is extensively used as affinity reagents in many applications in research, clinical diagnostics and therapy. With the ability to bind their ligands with high affinity and in a selective manner they are of great importance, and are by far the most commonly used affinity reagents. As when employing any affinity reagent the method used for detection needs to be considered. The antibodies' excellent capacities to bind their ligands need to be combined with high selectivity in order to be of great use. As a consequence, techniques for labeling of antibodies necessarily need to be of high quality. To make detection of the antibody and its binding events possible, a reporter group is normally attached to the antibody. Methods most commonly used when conjugating reporter groups to the antibody are based on the exploration of amine- or carboxyl-groups in the protein for coupling. Another application that ordinarily takes advantage of the surface-exposed functional groups in the protein, is the immobilization to a solid support. When applying amine- or carboxyl-based chemistry, normally a high degree of labeling or immobilization is obtained, but unfortunately the binding site might be compromised since the control of level and location of the labeling/coupling is limited. This means that optimization of the protocol is needed for every antibody and every conjugation/immobilization. Hence, a method for labeling, where specific and controlled conjugation can be achieved, would be a great advantage. To obtain specific conjugation, molecules that have a natural, specific and defined binding to antibodies could be employed.
Several Ig-binding molecules have been reported in literature. Among these, staphylococcal protein A (SpA), binding to VH and Fc, is one of the best characterized. Staphylococcal protein A is frequently used in many different applications, such as affinity chromatography, where its ability to bind antibodies is utilized. The protein is both used for purification of IgG molecules and as affinity tag for protein purification. The five homologous domains, EDABC, that constitute protein A, each consists of approximately 58 aa, and share the Ig-binding feature (Uhlén, Guss et al. 1984). NMR analysis of the structure of the B domain shows a three-helix bundle with the helices ordered in an anti-parallel fashion. The domains of protein A exhibit binding both to the Fc and the Fab regions of immunoglobulins. By X-ray crystallography the structure of the B domain in complex with the Fc region of IgG has been solved, and it revealed an interaction that mostly involved amino acids of hydrophobic character (Deisenhofer 1981). Binding seems to occur in the interface between CH2 and CH3 of IgG, where 11 residues from helix 1 and helix 2 of the B domain are suggested to participate (Deisenhofer 1981; Gouda, Shiraishi et al. 1997; Gouda, Shiraishi et al. 1998). Also, the structure of the interaction between protein A and the Fab region has been solved. The crystal structure of the D domain binding the Fab region of human IgM disclosed the involvement of 11 residues of helices 2 and 3 from the D domain, and an interaction of a polar character with the variable heavy chain (Graille, Stura et al. 2000). By altering two positions in the B-domain of protein A an engineered variant called the Z domain has been made. In the N-terminal of the Z domain an alanine residue was replaced by valine. A glycine-to-alanine substitution was made for the removal of a hydroxylamine cleavage site (Nilsson, Moks et al. 1987), which also resulted in loss of binding to the Fab region (Jansson, Uhlén et al. 1998; Graille, Stura et al. 2000). The Z domain is small (6.7 kDa), easy to produce, has a stable three dimensional structure and also the capacity to refold (Ståhl and Nygren 1997). It has earlier been proven to be suitable for chemical peptide synthesis and thereby the introduction of synthetic active groups has been possible, extending the usability of the domain (Engfeldt, Renberg et al. 2005).
Benzoylphenylalanine (BPA) is a synthetic amino acid that can be incorporated in a peptide during synthesis. Benzophenone (BP), which is part of BPA, is a photoreactive group that forms covalent bonds to other amino acids upon UV-exposure. BPA is considered to be efficient, stable and also easy to handle (Dorman and Prestwich 2000) and it is primarily used when mapping protein-ligand interactions. When mapping interactions the strategy is to produce variants of a protein with BPA incorporated at different positions, and then allow the protein to bind its interaction partner (Kawamura, Hindi et al. 2008). When subjecting the complex to UV light BPA forms a diradical, which renders the generation of a covalent bond between the protein and its interaction partner possible.